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WO2018219998A1 - Miarn en tant que biomarqueurs pour un syndrome de réponse inflammatoire systémique - Google Patents

Miarn en tant que biomarqueurs pour un syndrome de réponse inflammatoire systémique Download PDF

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Publication number
WO2018219998A1
WO2018219998A1 PCT/EP2018/064162 EP2018064162W WO2018219998A1 WO 2018219998 A1 WO2018219998 A1 WO 2018219998A1 EP 2018064162 W EP2018064162 W EP 2018064162W WO 2018219998 A1 WO2018219998 A1 WO 2018219998A1
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Prior art keywords
seq
sirs
mirna
individual
level
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Inventor
Andreas Keller
Susanne Schmolke
Cord Friedrich Stähler
Eckart Meese
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Siemens AG
Siemens Corp
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Siemens AG
Siemens Corp
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Publication of WO2018219998A1 publication Critical patent/WO2018219998A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • MIRNAS AS BIOMARKERS FOR A SYSTEMIC INFLAMMATORY RESPONSE SYNDROME
  • the present invention relates to a method of diagnosing an individual as having/suffering from a systemic
  • the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual. Furthermore, the present invention relates to a method of prognosing a systemic inflammatory response syndrome (SIRS) in an individual. In addition, the present invention relates to a kit for conducting the above-mentioned methods . BACKGROUND OF THE INVENTION
  • the reaction of the human body on a certain insult may result in severe consequences. Especially if the reaction is systemic, it may cause death of patients in short time periods.
  • the detection of systemic reactions of the human body to the external stimulus is the key for improved patient care .
  • SIRS systemic inflammatory response syndrome
  • Sepsis is one of the oldest and most pressing problems in medicine . Even with treatment in modern intensive care units ( ICUs ) , mortality rates of patients with sepsis are high . In Germany, 150.000 patients a year suffer from sepsis , over 50.000 patients die from the disease, making sepsis to one of the most common causes of death in Germany almost reaching heart failure as most common cause of death . For other developed countries the numbers are similar, the incidence of sepsis in the United States is reaching e.g. 0.3% per year .
  • PCR polymerase chain reaction
  • Lactate is re-measured if the initial lactate was elevated.
  • it is essential to diagnose or exclude any source of infection that would require emergent source control, such as necrotizing soft tissue infection, infection causing inflammation of the abdominal cavity lining, infection of the bile duct, or intestinal infarction.
  • a pierced internal organ free air on abdominal x-ray or CT scan) ; an abnormal chest x-ray
  • Biomarker might help to diagnose and/or predict SIRS, in particular sepsis, fast, flexible and reliable.
  • the present inventors studied the role of miRNAs as key modulators of the immune system. They found that miRNAs measured in biological samples, preferably in body fluids, more preferably in blood cells, reflect early and very specific immune responses against human pathologies. They considered this observation as an indication to further study the role of miRNAs in SIRS, e.g. sepsis. In particular, they analysed miRNA expression profiles of patients prior to surgical interventions and followed up the respective
  • SIRS SIRS without infection
  • SIRS with infection SIRS with infection
  • the miRNA level slightly changed pre and post surgical intervention . This might be caused by the fact that the immune system is specifically challenged and stressed during surgical intervention, which is also apparent on the miRNA level .
  • the inventors of the present invention determined miRNAs as biomarkers that allow to determine SIRS, in particular sepsis , with high reliability .
  • the present inventors identified miRNAs that are already dysregulated prior to surgical intervention. These miRNAs are suitable as predictive biomarkers of SIRS, in particular sepsis. Such biomarkers are not known yet.
  • the present inventors identified a specific blood cell sample type, preferably a leukocyte sample type, more preferably a lymphocyte sample type, as a special source of miRNAs having high diagnostic power.
  • the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the step of:
  • the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual (having SIRS) comprising the step of:
  • the present invention relates to a method of prognosing a systemic inflammatory response
  • SIRS syndrome in an individual comprising the step of:
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • primer pair for detecting at least one miRNA in a biological sample isolated from an individual (suspected of having SIRS) for diagnosing the individual as
  • SIRS systemic inflammatory response syndrome
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25.
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • primer pair for detecting at least one miRNA in a biological sample isolated from an individual (having SIRS) for determining the severity of a systemic inflammatory response syndrome (SIRS) in the individual,
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25.
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • SIRS systemic inflammatory response syndrome
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32.
  • the present invention relates to a kit comprising:
  • microRNA or “miRNA”, as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked
  • the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences
  • the miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs) .
  • the genes encoding miRNAs are longer than the processed mature miRNA molecules.
  • the miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70 nucleotide stem- loop structures known as pre-miRNAs in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha.
  • RNA-induced silencing complex RISC
  • Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC.
  • This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5' end.
  • the remaining strand known as the miRNA*, anti-guide (anti-strand), or passenger strand, is degraded as a RISC substrate.
  • the miRNA*s are derived from the same hairpin structure like the "normal” miRNAs. So if the "normal” miRNA is then later called the “mature miRNA” or “guide strand”, the miRNA* is the “anti- guide strand” or “passenger strand”.
  • microRNA* or “miRNA*”, as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked
  • the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 18 to 23 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences (e.g. biotin stretches) .
  • miRNA*s also known as the "anti-guide strands” or “passenger strands" are mostly complementary to the “mature miRNAs” or “guide strands”, but have usually single-stranded overhangs on each end. There are usually one or more mispairs and there are sometimes extra or missing bases causing single-stranded “bubbles”.
  • the miRNA*s are likely to act in a regulatory fashion as the miRNAs (see also above) .
  • the terms “miRNA” and “miRNA*” are interchangeable used.
  • miRBase refers to a well established repository of validated miRNAs. The miRBase
  • miRNA sequences are a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database) , with information on the location and sequence of the mature miRNA sequence
  • sequences of the miRNAs described herein are based on miRBase version 21.
  • nucleotides refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals:
  • nucleosides refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH) , producing nucleotides, which are the molecular building blocks of DNA and RNA.
  • -CH2-OH primary alcohol group
  • polynucleotide means a molecule of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together.
  • the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally spacer elements and/or elongation elements described below.
  • the depiction of a single strand of a polynucleotide also defines the sequence of the complementary strand.
  • Polynucleotides may be single stranded or double stranded, or may contain
  • polynucleotide means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands.
  • the polynucleotide may be DNA, both cDNA and genomic DNA, RNA, cRNA or a hybrid, where the polynucleotide sequence may contain combinations of deoxyribonucleotide or
  • ribonucleotide bases and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.
  • Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods .
  • a polynucleotide as a single polynucleotide strand provides a probe (e.g. miRNA capture probe) that is capable of binding to, hybridizing with, or detecting a target of complementary sequence, such as a nucleotide sequence of a miRNA or miRNA*, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe e.g. miRNA capture probe
  • a target of complementary sequence such as a nucleotide sequence of a miRNA or miRNA*
  • Polynucleotides in their function as probes may bind target sequences, such as nucleotide sequences of miRNAs or miRNAs*, lacking complete complementarity with the polynucleotide sequences depending upon the stringency of the hybridization condition. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence, such as a nucleotide sequence of a miRNA or miRNA*, and the single stranded polynucleotide described herein.
  • polynucleotide variants including
  • polynucleotide fragments or polynucleotide mutants and the miRNA variants including miRNA fragments or miRNA mutants are further defined below. Described herein are polynucleotides in form of single polynucleotide strands as probes for binding to, hybridizing with or detecting complementary sequences of miRNAs (targets) , which are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32.
  • targets selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32.
  • the polynucleotide e.g.
  • the polynucleotide used as a probe for detecting a miRNA or miRNA* may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind.
  • the polynucleotide e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may also be modified, e.g. may comprise an
  • elongation element For use in a RAKE or MPEA assay, a polynucleotide with an elongation element may be used as a probe.
  • the elongation element comprises a nucleotide sequence with 1 to 30 nucleotides chosen on the basis of showing low complementarity to potential target sequences, such as nucleotide sequences of miRNAs or miRNAs*, therefore
  • the polynucleotide e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may be present in form of a tandem, i.e.
  • Said polynucleotide hybrid/tandem may comprise a spacer element.
  • the polynucleotide hybrid/tandem as a probe may comprise a spacer (SP) element.
  • SP spacer
  • n 0, i.e. that there is no spacer between the two miRNA sequence stretches.
  • label means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA) , biotin, digoxigenin, or haptens and other entities which can be made detectable.
  • a label may be incorporated into nucleic acids at any position, e.g. at the 3' or 5' end or internally.
  • the polynucleotide for detecting a miRNA include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA) , biotin, digoxigenin, or haptens and other entities which can be made detectable.
  • a label may be incorporated into nucleic acids at any position, e.g. at the 3' or 5' end or internally.
  • polynucleotide probe and/or the miRNA itself may be
  • stringent hybridization conditions means conditions under which a first nucleic acid sequence (e.g. polynucleotide in its function as a probe for detecting a miRNA or miRNA*) will hybridize to a second nucleic acid sequence (e.g. target sequence such as
  • nucleotide sequence of a miRNA or miRNA* such as in a complex mixture of nucleic acids.
  • Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • the Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 tol.O M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20°C for short probes (e.g., about 10-35 nucleotides) and up to 60°C for long probes (e.g., greater than about 50
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a positive signal may be at least 2 to 10 times background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense" strand.
  • Residues in two or more polynucleotide s are said to "correspond" to each other if the residues occupy an
  • analogous position in the polynucleotide structures It is well known in the art that analogous positions in two or more polynucleotides can be determined by aligning the
  • polynucleotide sequences based on nucleic acid sequence or structural similarities.
  • alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW (see www .ebi.ac.uk/clustalw) or Align (see
  • systemic inflammatory response syndrome systemic inflammatory response syndrome
  • SIRS systemic inflammatory response to a variety of severe clinical insults.
  • the causes of SIRS are broadly classified as infectious or non ⁇ infectious.
  • the causes of SIRS include, but are not limited to, infection, trauma, burns, pancreatitis, ischemia, hemorrhage, and others.
  • the other causes of SIRS include, but are not limited to, complications of surgery, adrenal
  • the syndrome is manifested by more than one of the following conditions: (i) body temperature less than 36 C or greater than 38°C, (ii) heart rate greater than 90 beats per minute, (iii) tachypnea (high respiratory rate) , with greater than 20 breaths per minute; or, an arterial partial pressure of
  • sepsis If signs of an infection are present, the syndrome is termed sepsis .
  • SIRS may be designated as SIRS with infection (sepsis) or SIRS without infection.
  • SIRS may be termed SIRS dependent on/caused by infection (sepsis) or SIRS independent of/not caused by infection.
  • infection refers to a microbial phenomenon characterized by an inflammatory
  • the infection may be caused by bacteria, fungi,
  • bacteremia refers to the presence of (viable) bacteria in the blood.
  • viremia refers to a condition where (viable) viruses enter the bloodstream and hence have access to the rest of the body.
  • fungemia refers to the presence of
  • parasitemia refers to a condition where (viable) parasites enter the bloodstream and hence have access to the rest of the body.
  • SIRS infectious disease
  • sepsis refers to the systemic response to infection. In association with infection, manifestations of sepsis are the same as those described for SIRS, and include more than one of the following conditions:
  • tachypnea high respiratory rate
  • sepsis is severe sepsis, more preferably septic shock .
  • severe sepsis refers to sepsis associated with organ dysfunction, hypoperfusion abnormality, or sepsis-induced hypotension.
  • Hypoperfusion abnormalities include, but are not limited to, lactic
  • hypotension is defined by the presence of a systolic blood pressure of less than 90 mm Hg or its reduction by 40 mm Hg or more from baseline in the absence of other causes for hypotension (e.g. cardiogenic shock) .
  • septic shock refers to a subset of severe sepsis and is defined as sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction.
  • Perfusion abnormalities include, but are not limited to, lactic acidosis, oliguria, or an acute alternation in mental status. In this respect, it should be noted that patients who are receiving inotropic or
  • vasopressor agents may not be hypotensive at the time that perfusion abnormalities are measured.
  • SIRS systemic inflammatory response syndrome
  • individual may be diagnosed as suffering from SIRS without infection or SIRS with infection (sepsis) , or as not
  • SIRS inflammatory response syndrome
  • the sepsis is severe sepsis. Even more
  • the sepsis is septic shock.
  • SIRS systemic inflammatory response syndrome
  • SIRS means determining whether an
  • SIRS system for remission
  • the term "individual”, as used herein, refers to any subject for whom it is desired to know whether she or he suffers from SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • the term "individual”, as used herein, refers to a subject suspected to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • the individual may be diagnosed to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. diseased, or may be diagnosed to be not affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. healthy with respect to SIRS.
  • the term "individual”, as used herein, also refers to a subject that is affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e. diseased.
  • the individual may be retested for SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, and may be diagnosed to be still affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, i.e.
  • SIRS diseased, or not affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, anymore, i.e. healthy with respect to SIRS, for example after therapeutic intervention.
  • the individual may further be retested for SIRS and may be diagnosed as having developed an advanced form of SIRS, in particular sepsis, or as suffering from a serious form of SIRS, in particular sepsis, such as severe sepsis or septic shock.
  • the term "individual”, as used herein, further refers to any subject for whom it is desired to know whether she or he may develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • SIRS in particular SIRS with infection (sepsis) or SIRS without infection.
  • “individual”, as used herein, refers to a subject who has a predisposition to develop SIRS or will likely develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • the individual may be prognosed to develop SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, as the present inventors surprisingly found that miRNAs representative for SIRS, in particular SIRS with infection (sepsis) or SIRS without infection, are already present/deregulated in a biological sample before SIRS occurs or during the early stage of SIRS.
  • the term "individual”, as used herein, preferably refers to a subject who has experienced, experiences, or will experience an event involving a risk of developing SIRS, in particular SIRS with infection (sepsis) or SIRS without infection. This event may have caused, causes, or will likely cause SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • the term "event involving a risk of developing SIRS”, as used herein, encompasses any incident that may be responsible for the development of SIRS or a trigger of SIRS.
  • the event involving a risk of developing SIRS encompasses a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical
  • SIRS may be SIRS with infection (sepsis) or SIRS without infection.
  • the individual may suffer from a disease that requires a surgery, suffer from an infection, experience a trauma, have burns, suffer from pancreatitis, suffer from ischemia, have a tissue injury, have a hemorrhagic shock, and/or have an immune-mediated organ injury.
  • the individual may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human subjects as individuals are
  • control subject refers to a subject known to be affected by SIRS, in particular SIRS with infection (sepsis) or SIRS without infection (positive control), i.e. diseased.
  • control subject also refers to a subject known to be not
  • SIRS disease 2019
  • SIRS with infection infection
  • SIRS without infection negative control
  • control subject preferably refers to a subject not suffering from SIRS who has
  • the event involving a risk of developing SIRS is preferably a surgical
  • the (control) subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey.
  • Human (control) subjects as
  • treatment in particular "therapeutic
  • treatment refers to any therapy which improves the health status and/or prolongs (increases) the lifespan of an individual.
  • Said therapy may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or
  • the disease may be SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • SIRS in particular SIRS with infection (sepsis) or SIRS without infection.
  • the therapeutic treatment of SIRS includes, but is not limited to, antimicrobial therapy, fluid therapy, vasopressor therapy, inotropic therapy, and corticosteroid therapy .
  • the term "biological sample”, as used herein, refers to any biological sample from an individual or a (control) subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32.
  • the biological sample may be a body fluid sample or a tissue sample.
  • biological samples encompassed by the present invention are tissue samples, blood (e.g.
  • a sample may be a mixture of a blood sample and a urine sample.
  • Said biological samples may be provided by removing a biological sample from an individual or (control) subject, but may also be provided by using a previously isolated sample.
  • a blood sample may be taken from an individual or (control) subject by conventional blood collection techniques, or a tissue sample may be taken from an individual or (control) subject by biopsy.
  • the biological sample e.g. urine sample, blood sample or tissue sample, may be obtained from an individual or (control) subject prior to the initiation of a therapeutic treatment, during the
  • the biological sample is obtained from at least one (control) subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1.000 (control) subject(s), it is designated as "reference biological sample".
  • the reference biological sample is from the same source than the biological sample of the individual to be tested, e.g. both are blood samples, urine samples or tissue samples. It is further preferred that both are from the same species, e.g. from a human. It is also (alternatively or additionally) preferred that the measurements of the reference biological sample of the (control) subject and the biological sample of the individual to be tested are identical, e.g. both have an identical volume. It is particularly preferred that the reference biological sample and the biological sample are from (control) subjects/individuals of the same sex and similar age.
  • body fluid sample refers to any liquid sample derived from the body of an individual or (control) subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32.
  • body fluid sample refers to any body fluid sample from an individual or
  • control subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO. 32.
  • Said body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.
  • body fluid sample also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions.
  • the body fluid samples may be mixed or pooled.
  • a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample.
  • Said body fluid sample may be provided by removing a body liquid from an individual or (control) subject, but may also be provided by using previously isolated body fluid sample material.
  • the body fluid sample allows for a non ⁇ invasive analysis of an individual. It is further preferred that the body fluid sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml, and most preferably of between 1 and 5 ml . If the body fluid sample is obtained from at least one (control subject), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1.000 control subject(s), it is designated as "reference body fluid sample”.
  • blood sample encompasses a whole blood sample or a blood fraction sample such as a blood cell/cellular fraction, blood serum, or blood plasma sample. It is preferred that the blood serum or plasma sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml and most preferably of between 1 and 5 ml.
  • blood cell/cellular fraction refers to a blood cell/cellular portion which has been produced from whole blood by removing the extracellular fraction (serum and/or plasma) .
  • the blood cell/cellular fraction is depleted of the extracellular blood components, such as serum and/or plasma.
  • the blood sample is a blood
  • the blood cell/cellular fraction comprises/consists of erythrocytes, leukocytes, and thrombocytes .
  • the blood sample is a blood cell sample.
  • the blood cell sample is a sample of leukocytes. More preferably, the blood cell sample is a sample of lymphocytes.
  • level refers to an amount (measured for example in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration) of the miRNA(s) described herein, in particular of the miRNA(s) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO : 32.
  • concentration e.g. absolute or relative concentration
  • level also comprises scaled, normalized, or scaled and normalized amounts or values .
  • the level determined herein is the expression level .
  • sensitivity refers to the number of true positive patients (%) with regard to the number of all patients (100%) .
  • the individuals may be
  • SIRS subjects having SIRS, in particular SIRS with infection
  • Specificity relates to the number of true negative individuals (%) with regard to the number of all healthy subjects (100%) .
  • accuracy means a statistical measure for the correctness of classification or identification of sample types. The accuracy is the
  • the result of each analysis group is usually calculated from a plurality of isolated samples, i.e. from at least 2 isolated samples, preferably from between 2 and 20, more preferably from between 10 and 60, and even more preferably from between 50 and 100 isolated samples, e.g. selected from the group consisting of subjects not suffering from SIRS, in particular SIRS with infection and/or SIRS without infection, and subjects having SIRS, in particular SIRS with infection (sepsis) or SIRS without infection.
  • the methods of the present invention can be carried out in combination with other methods for diagnosing an individual as
  • SIRS having/suffering from SIRS or not, for prognosing SIRS in an individual or for determination the severity of SIRS in an individual to increase the overall sensitivity and/or
  • the determination of the level of the miRNA(s) of the present invention allows the diagnosis of SIRS in an individual, the prognosis of SIRS in an individual, or the determination of the severity of SIRS in an individual.
  • AUC as used herein, relates to an
  • ROC Receiver Operating Characteristic
  • ROC curve is a measure for the accuracy of a diagnostic test (the larger the area the better, optimum is 1, a random test would have a ROC curve lying on the diagonal with an area of 0.5 (see, for reference, for example, JP . Egan.
  • kit of parts (in short: kit)" is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components. Said kit may allow point-of-care testing (POCT) .
  • POCT point-of-care testing
  • POCT point-of-care testing
  • Point-of-care tests are simple medical tests that can be performed at the bedside.
  • the driving notion behind POCT is to bring the test conveniently and immediately to the individual to be tested. This increases the likelihood that the individual, physician, and care team will receive the results quicker, which allows for immediate clinical management decisions to be made.
  • POCT is often accomplished through the use of transportable, portable, and handheld instruments and test kits. Small bench analyzers or fixed equipment can also be used when a handheld device is not available - the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the individual so that the treatment plan can be adjusted as necessary before the individual leaves the hospital .
  • the present inventors studied the role of miRNAs as key modulators of the immune system. They found that miRNAs measured in biological samples, preferably in body fluids, more preferably in blood cells, reflect early and very specific immune responses against human pathologies. They considered this observation as an indication to further study the role of miRNAs in SIRS, e.g. sepsis. In particular, they analysed miRNA expression profiles of patients prior to surgical interventions and followed up the respective
  • SIRS SIRS without infection
  • SIRS with infection SIRS with infection
  • the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the step of: determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • miRNA(s)) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual.
  • the method may be performed after an event involving a risk of an individual of developing SIRS, or before and after an event involving a risk of an individual of developing SIRS.
  • the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,
  • hemorrhagic shock immune-mediated organ injury, or any combination thereof.
  • SIRS may be SIRS with infection (sepsis) or SIRS without infection. Sepsis is preferably severe sepsis, more
  • the infection may be selected from the group consisting of bacteremia, viremia, fungemia, and parasitemia .
  • the level of the at least one miRNA e.g. 1,
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto. More preferably, the level of the at least one miRNA (e.g. 1, 2,
  • the level of the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 and a sequence having at least 80% sequence identity thereto.
  • Said at least one miRNA allows to determine whether the individual suffers from SIRS. Said at least one miRNA, however, does not allow to determine whether SIRS is SIRS with infection
  • SIRS sepsis
  • the at least one miRNA e.g. 1, 2, 3, 4,
  • miRNA(s) which is preferably selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS with infection (sepsis) .
  • the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS without infection.
  • the at least one miRNA e.g. 1, 2, 3, 4, 5,
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. More preferably, the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. In particular, said at least one miRNA allows to determine whether the individual does not have SIRS (SIRS with infection (sepsis) and/or SIRS without infection).
  • SIRS SIRS with infection
  • SIRS SIRS without infection
  • the level of the at least one miRNA is compared to a reference level of said at least one miRNA.
  • the present invention relates to a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the steps of:
  • determining the level of at least one miRNA e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • miRNA ( s ) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more
  • the reference level may be any level which allows to determine whether an individual suffers from SIRS or not. It may be obtained from a (control) subject (i.e. a subject different from the individual to be tested/diagnosed) or from the same individual.
  • the reference level is the level determined by measuring at least one reference
  • biological sample e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject not suffering from SIRS, in particular not suffering from SIRS with
  • infection and/or SIRS without infection
  • the at least one subject not suffering from SIRS can be considered as being healthy with respect to SIRS.
  • the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS.
  • the at least one subject not suffering from SIRS is a subject who has experienced an event involving a risk of developing SIRS without developing SIRS.
  • the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,
  • the infection may be a bacterial, viral, fungal, and/or parasitic infection. It is more preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery. It is particularly preferred that the reference level is the level taken from the subject after the experience of an event involving a risk of developing SIRS.
  • reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested.
  • Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof.
  • the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed.
  • the level of the at least one miRNA is compared to a reference level of said at least one miRNA.
  • Said reference level is the level determined by measuring a reference biological sample. For example, if the level of the miRNA according to SEQ ID NO: 1 is determined in a biological sample from an individual , it is compared to a reference level of the miRNA according to SEQ ID NO : 1 determined in a reference biological sample . Alternatively, if the level of the miRNA according to SEQ ID NO : 1 and the level of the miRNA according to SEQ ID NO : 2 is determined in a biological samp1e from an individual , both levels are compared to the respective reference levels , i.e.
  • the level of the miRNA according to SEQ ID NO : 1 is compared to the reference level of the miRNA according to SEQ ID NO : 1 and the level of the miRNA according to SEQ ID NO : 2 is compared to the reference level of the miRNA according to SEQ ID NO : 2 determined in a reference biological sample .
  • the level of the at least one miRNA e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s) selected from the group
  • SEQ ID NO: 1 consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the
  • the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS, and/or
  • the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80%
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.
  • the term "comparable” means that the level may vary between 0 and 10%, e.g. 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.
  • both levels are identical with each other (i.e. 0% variation) .
  • the term "comparable” in this respect also means that the detected level variation may be within the accuracy of a measurement.
  • the accuracy of a measurement depends on the measurement method used. In case it is determined whether the individual does not have SIRS, the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .
  • the reference level is the level determined by measuring a reference biological sample isolated from the individual before an event involving a risk of developing SIRS, and wherein the biological sample is isolated from the individual after the event involving a risk of developing SIRS.
  • the reference biological reference sample may be isolated from the individual 1, 2, 3, 4, 5, or 6 day(s) before an event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) before an event involving a risk of developing SIRS, or immediately before an event involving a risk of developing SIRS.
  • the reference biological sample is isolated from the individual between 6 days and 1 hour, more preferably between 2 days and 1 hour, and even more preferably between 1 day and 1 hour before an event involving a risk of developing SIRS.
  • the biological sample may be isolated from the individual immediately after the event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) after the event
  • the biological sample is isolated from the individual between 1 hour and 6 days, more preferably between 1 hour and 4 days, and even more preferably between 1 hour and 2 days after the event involving a risk of developing SIRS .
  • reference biological sample isolated from the individual before an event involving a risk of developing SIRS and the biological sample isolated from the individual after the event involving a risk of developing SIRS are from the same source (e.g. blood samples) .
  • the event involving a risk of developing SIRS is preferably a surgical intervention/surgery.
  • the level of the at least one miRNA e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s) selected from the group
  • SEQ ID NO: 1 consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the
  • the level of the at least one miRNA (e.g. 1, 2, 3, 4, or 5 miRNA(s)) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS, and/or
  • the level of the at least one miRNA (e.g. 1, 2, 3, or 4 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the at least one miRNA (e.g. 1, 2, 3, or 4 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the
  • the level of the at least one miRNA (e.g. 1 or 2 miRNA(s)) selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the individual does not have SIRS.
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold
  • the level of the at. least one miRNA is: at. least 0 6-fold, at. least 0.7- fold, at least 0 .8 -fold, at least 0. 9 -fold, at least 1 .0- fold, at least 1 .1 -fold, at least 1. 2 -fold, at least 1 .3- fold, at least 1 .4 -fold, at least 1. 5 -fold, at least 1 .6- fold, at least 1 .7 -fold, at least 1. 8 -fold, at least 1 .9- fold, at least 2 .0 -fold, at least 2. 1 -fold, at least 2 .2- fold, at least 2 .3 -fold, at least 2.
  • the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .
  • SIRS is selected from the group consisting of SIRS with infection (sepsis) and SIRS without infection.
  • the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, or 9 miRNA(s)) is selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto.
  • Said at least one miRNA allows to determine whether the individual suffers from SIRS with infection (sepsis) or SIRS without infection .
  • SIRS is SIRS with infection (sepsis) and the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%
  • the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the
  • SIRS with infection saliva
  • SIRS is SIRS without infection and the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto.
  • miRNA e.g. 1, 2, or 3 miRNA(s)
  • the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the individual has SIRS without infection.
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.
  • the method of the first aspect of the present invention is formulated as follows: a method of diagnosing an individual (suspected of having SIRS) as having/suffering from a systemic inflammatory response syndrome (SIRS) or not comprising the steps of:
  • determining the level of at least one miRNA e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 miRNA ( s ) ) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more miRNAs (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 miRNA ( s ) ) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more miRNA ( s )
  • the reference biological reference sample may be any suitable biological reference sample.
  • the reference biological sample is isolated from the individual 1, 2, 3, 4, 5, or 6 day(s) before an event involving a risk of developing SIRS, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hour(s) before an event involving a risk of developing SIRS, or immediately before an event involving a risk of developing SIRS.
  • the reference biological sample is isolated from the individual between 6 days and 1 hour, more preferably between 2 days and 1 hour, and even more preferably between 1 day and 1 hour before an event involving a risk of developing SIRS.
  • the biological sample may be isolated from the
  • the biological sample is isolated from the individual between 1 hour and 6 days, more preferably between 1 hour and 4 days, and even more preferably between 1 hour and 2 days after the event involving a risk of developing SIRS .
  • the reference biological sample isolated from the individual before an event involving a risk of developing SIRS and the biological sample isolated from the individual after the event involving a risk of developing SIRS are from the same source (e.g. blood samples) .
  • SIRS may be SIRS with infection (sepsis) or SIRS without infection. Sepsis is preferably severe sepsis, more
  • the infection may be selected from the group consisting of bacteremia, viremia, fungemia, and parasitemia .
  • the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 miRNA ( s ) ) is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto. More preferably, the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, and a sequence having at least 80% sequence identity thereto.
  • the level of the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, and a sequence having at least 80% sequence identity thereto.
  • Said at least one miRNA allows to determine whether the individual suffers from SIRS.
  • Said at least one miRNA does not allow to determine whether SIRS is SIRS with infection
  • SIRS sepsis
  • the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS with infection (sepsis) .
  • the at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) which is preferably selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto allows to determine whether the individual has SIRS without infection.
  • the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, or 8 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto. More preferably, the at least one miRNA (e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)) is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto.
  • said at least one miRNA allows to determine whether the individual does not have SIRS (SIRS with
  • the level of the at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 miRNA(s)) selected from the group
  • step (i) consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual has SIRS, and/or
  • the level of the at least one miRNA (e.g. 1, 2, 3, 4, or 5 miRNA(s)) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the
  • the level of the at least one miRNA (e.g. 1, 2, 3, or 4 miRNA(s)) selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the individual does not have SIRS, and/or
  • the level of the at least one miRNA (e.g. 1 or 2 miRNA(s)) selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, and a sequence having at least 80% sequence identity thereto is determined in steps (i) and (ii) , wherein the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual does not have SIRS.
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold below/above the reference level.
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1.0- fold, at least 1.1-fold, at least 1.2-fold, at least 1.3- fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5- fold, at least 2.6-fold, at least 2.7-fold, at least 2.8- fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.
  • the above method may also refer to a method of determining the absence of SIRS in an individual (suspected of suffering from SIRS) .
  • the method is a method of diagnosing an individual as having/suffering from SIRS with infection
  • determining the level of at least one miRNA e.g. 1, 2, 3, 4, 5, or 6 miRNA(s) selected from the group
  • SEQ ID NO: 9 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, in particular SIRS with infection (sepsis) ,
  • the level of said at least one miRNA determined in step (ii) being above the level determined in step (i) indicates that the individual has SIRS with
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold above the reference level.
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8- fold, at least 0.9-fold, at least 1.0-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4- fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3- fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9- fold, or at least 3.0-fold above the reference level.
  • the method is also a method of
  • diagnosing an individual has having /suffering from SIRS without infection.
  • the method of diagnosing an individual has having /suffering from SIRS without infection.
  • having/suffering from SIRS without infection comprises the steps of: (i) determining the level of at least one miRNA (e.g. 1, 2, or 3 miRNA(s)) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, in particular SIRS without infection, (ii) determining the level of the at least one miRNA in a miRNA (e.g. 1, 2, or 3 miRNA(s)) selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, and a sequence having at least 80% sequence identity thereto in a biological sample isolated from an individual before an event involving a risk of developing SIRS, in particular SIRS without infection, (ii) determining the level of the at least one miRNA in a
  • miRNA e.g. 1, 2, or 3 miRNA(s)
  • the level of said at least one miRNA determined in step (ii) being below the level determined in step (i) indicates that the individual has SIRS without infection .
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold below the reference level.
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0. fold, at least 0. 9 -fold, at least 1. 0 -fold, at least 1. 1- fold, at least 1. 2 -fold, at least 1. 3 -fold, at least 1. 4- fold, at least 1. 5 -fold, at least 1. 6 -fold, at least 1. 7- fold, at least 1. 8 -fold, at least 1. 9 -fold, at least 2.
  • SIRS systemic inflammatory response syndrome
  • the present invention relates to a method of determining the severity of a systemic
  • SIRS inflammatory response syndrome
  • determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample
  • miRNA e.g. 1, 2, 3, 4, 5, or 6 miRNA(s)
  • the level of the at least one miRNA is compared to a reference level of said at least one miRNA.
  • the present invention relates to a method of determining the severity of a systemic inflammatory response syndrome (SIRS) in an individual
  • determining the level of at least one miRNA e.g. 1, 2, 3, 4, 5, or 6 miRNA(s) selected from the group
  • SEQ ID NO: 14 SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in a biological sample isolated from an individual, and (ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA.
  • the reference level may be any level which allows to determine the severity of SIRS in the individual.
  • the reference level is the level determined by measuring at least one reference
  • biological sample e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200,
  • infection and/or SIRS without infection
  • the at least one subject not suffering from SIRS can be considered as being healthy with respect to SIRS.
  • the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS.
  • the at least one subject not suffering from SIRS is a subject who has experienced an event involving a risk of developing SIRS without developing SIRS. It is preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery, an infection, e.g. an infection caused by a surgical intervention/surgery, a trauma, burns, pancreatitis, ischemia, tissue injury,
  • the infection may be a bacterial, viral, fungal, and/or parasitic infection. It is more preferred that the event involving a risk of developing SIRS is a surgical intervention/surgery. It is particularly preferred that the reference level is the level taken from the subject after the experience of an event involving a risk of developing SIRS.
  • the reference level is the level determined by measuring at least one reference biological sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject suffering from SIRS without infection, e.g.
  • the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects suffering from SIRS without infection. It is even more preferred that the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects suffering from SIRS without infection. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects suffering from SIRS without infection .
  • reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested.
  • Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof.
  • the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed.
  • the level of the at least one miRNA above the reference level indicates that the individual has a severe form of SIRS/has SIRS with infection (sepsis) .
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, more preferably at least 1.2-fold or 1.5-fold, and even more preferably at least 2.0-fold or 3.0-fold above the reference level.
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8- fold, at least 0.9-fold, at least 1.0-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4- fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3- fold, at least 2.4-fold, at least 2.5-fold, at least 2.6- fold, at least 2.7-fold, at least 2.8-fold, at least 2.9- fold, or at least 3.0-fold above the reference level.
  • Sepsis is preferably severe sepsis, more preferably septic shock.
  • Biomarkers in particular miRNA biomarkers, which allow to determine whether the individual will likely develop SIRS (predictive biomarkers, in particular predictive miRNA biomarkers) are not known yet.
  • SIRS predictive biomarkers, in particular predictive miRNA biomarkers
  • miRNAs that are already dysregulated prior to surgical intervention/surgery. These miRNAs allow to
  • the present invention relates to a method of prognosing/predicting a systemic inflammatory response syndrome (SIRS) in an individual comprising the step of:
  • determining the level of at least one miRNA e.g. 1, 2, 3, 4, 5, 6, or 7 miRNA(s) selected from the group consisting of
  • the level of the at least one miRNA is compared to a reference level of said at least one miRNA.
  • the present invention relates to a method of prognosing a systemic inflammatory response syndrome (SIRS) in an individual comprising the steps of: (i) determining the level of at least one miRNA (e.g. 1, 2, 3, 4, 5, 6, or 7 miRNA(s)) selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32 and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e.
  • SIRS systemic inflammatory response syndrome
  • the reference level may be any level which allows to determine the severity of SIRS in the individual.
  • the reference level is the level determined by measuring at least one reference
  • biological sample e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference biological sample(s), isolated from at least one (control) subject not suffering from SIRS, in particular not suffering from SIRS with infection (sepsis) and/or SIRS without infection, e.g.
  • the reference level is the level determined by measuring between 2 and 500 reference biological samples isolated from between 2 and 500 subjects not suffering from SIRS.
  • the reference level is determined by measuring between 50 and 500 reference biological samples isolated from between 50 and 500 subjects not suffering from SIRS. It is most preferred that the reference level is determined by measuring between 100 and 500 reference biological samples isolated from between 100 and 500 subjects not suffering from SIRS.
  • reference biological sample per subject for analysis. If additional reference biological samples are required, e.g. to determine the reference level in different reference biological samples, the same subject may be (re) tested.
  • Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the "average" value (e.g. mean, median or modal value) thereof.
  • the reference biological sample is from the same source (e.g. blood sample) than the biological sample isolated from the individual. It is further preferred that the reference level is obtained from a subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the individual to be tested or diagnosed.
  • the level of the at least one miRNA below the reference level indicates that the individual has a predisposition to develop SIRS or that the individual will likely develop SIRS.
  • the level of the at least one miRNA is at least 0.6-fold or 0.7-fold, preferably at least 0.8-fold or 0.9-fold, and more preferably at least 1.2-fold or 1.3-fold below the reference level.
  • the level of the at least one miRNA is at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1-fold, at least 1.2-fold, or at least 1.3-fold below the reference level.
  • the individual is a mammal, preferably a human.
  • biological sample is a body fluid or tissue sample.
  • the body fluid sample is selected from the group consisting of a blood sample, a urine sample, and a
  • the blood sample is a whole blood sample or a blood fraction sample. Even more preferably, the blood fraction sample is a blood
  • cell/cellular fraction sample a blood serum sample, or a blood plasma sample.
  • the blood cell/cellular fraction in one embodiment, the blood cell/cellular fraction
  • erythrocytes comprises/consists of erythrocytes, leukocytes, and
  • the blood sample is a blood cell sample of leukocytes, in particular a sample of
  • lymphocytes lymphocytes
  • the level of more than one miRNA is determined, e.g. of two or more miRNAs, it is referred herein to a set comprising at least two miRNAs.
  • the determination of the level of the at least one miRNA may be carried out by any convenient means for determining the level of a nucleotide sequence such as miRNA. For this purpose, qualitative, semi-quantitative and quantitative detection methods can be used. Quantitative detection methods are preferred. A variety of techniques are well known to the person skilled in the art. For example, the level of the at least one miRNA can be determined in the methods of the first to third aspect of the present invention by nucleic acid hybridization, nucleic acid amplification, polymerase
  • nucleic acid hybridization is performed using a
  • microarray/biochip or using in situ hybridization,
  • the nucleic acid amplification is performed using real- time PCR (RT-PCR) or quantitative real-time PCR (qPCR) ,
  • the sequencing is next generation sequencing, or
  • the immunochemical method is an enzyme linked
  • ELISA immunosorbent assay
  • Nucleic acid amplification may be any suitable nucleic acid amplification.
  • Nucleic acid amplification may be any suitable nucleic acid amplification.
  • RT-PCR real time polymerase chain reaction
  • RT qPCR real time quantitative PCR
  • the real time polymerase chain reaction (RT-PCR) may include the following steps: (i) extracting total RNA from the biological sample isolated from the individual, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using miRNA-specific primers, (iii) designing miRNA-specific cDNA forward primers and providing universal reverse primers to amplify the cDNA via polymerase chain reaction (PCR) , (iv) adding a
  • RT-PCR real time polymerase chain reaction
  • RT qPCR real time quantitative PCR
  • Nucleic acid hybridization may be any suitable nucleic acid hybridization.
  • Nucleic acid hybridization may be any suitable nucleic acid hybridization.
  • nucleic acid hybridization for example, the polynucleotides (probes) described herein with
  • microarray/biochip Said microarray/biochip is then incubated with miRNAs, isolated (e.g. extracted) from the biological sample, which may be labelled or unlabelled.
  • the success of hybridisation may be controlled and the intensity of hybridization may be determined via the hybridisation signal of the label in order to determine the level of each tested miRNA in said biological sample.
  • the miRNA level may be determined using an immunochemical method, e.g. using an ELISA.
  • Said method may include the following steps: (i) isolating miRNAs from a biological sample, (ii) hybridizing polynucleotide probes (complementary) to the miRNAs to obtain hybrids of said polynucleotides probes and said miRNAs, and (iii) binding said hybrids to antibodies capable of specifically binding hybrids of said polynucleotide probes and said miRNAs, and (iv) detecting the antibody-bound hybrids.
  • the level of the at least one miRNA is the expression level of said at least one miRNA.
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • primer pair for detecting at least one miRNA in a biological sample isolated from an individual (suspected of having SIRS) for diagnosing the individual as
  • SIRS systemic inflammatory response syndrome
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25.
  • At least one polynucleotide is preferred that at least one polynucleotide
  • probe/primer in particular primer pair
  • SIRS SIRS with infection
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, and SEQ ID NO: 25. It is alternatively preferred that at least one
  • polynucleotide is used for detecting at least one miRNA in a biological sample isolated from an individual for diagnosing the individual as having/suffering from SIRS without infection,
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 15.
  • the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or
  • the at least one polynucleotide has at least 80
  • the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .
  • the at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for the at least one polynucleotide (probe/primer, in particular primer pair) described above.
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • primer pair for detecting at least one miRNA in a biological sample isolated from an individual (having SIRS) for determining the severity of a systemic inflammatory response syndrome (SIRS) in the individual,
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25.
  • the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or
  • the at least one polynucleotide has at least 80 ⁇ 6 , more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
  • the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .
  • the at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for the at least one polynucleotide (probe/primer, in particular primer pair) described above.
  • the present invention relates to the use of at least one polynucleotide (probe/primer, in
  • primer pair for detecting at least one miRNA in a biological sample isolated from an individual for
  • SIRS systemic inflammatory response syndrome
  • the at least one miRNA is selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 32.
  • the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or
  • the at least one polynucleotide has at least 80 ⁇ 6 , more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the
  • the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i) .
  • the at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for the at least one polynucleotide (probe/primer, in particular primer pair) described above.
  • the present invention relates to a kit comprising:
  • SEQ ID NO: 9 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 25, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, i.e.
  • kit comprising the means referred to in
  • (ia) is for diagnosing an individual as having/suffering from a systemic inflammatory response syndrome (SIRS) or not,
  • (ib) is for determining the severity of a systemic
  • SIRS inflammatory response syndrome
  • (ic) is for prognosing/predicting a systemic inflammatory
  • SIRS response syndrome
  • kit is useful for conducting the methods according to the first to third aspect of the present invention.
  • kit comprising the means referred to in
  • the at least one reference may be any reference which allows to diagnose whether an individual suffers from SIRS or not, to determine the severity of SIRS in an individual, and/or to prognose/predict SIRS in an individual.
  • (ib) comprise at least one polynucleotide (probe/primer, in particular primer pair) as defined in the fifth aspect of the present invention, and/or
  • (ic) comprise at least one polynucleotide (probe/primer, in particular primer pair) as defined in the sixth aspect of the present invention.
  • the means in (ia) , (ib) , and/or (ic) further comprise at least one polynucleotide (probe) and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.
  • the means in (ia) , (ib) , and/or (ic) comprise at least one polynucleotide (probe) (see third to sixth aspect of the present invention) ,
  • At least one primer pair (see third to sixth aspect of the present invention), and/or
  • polynucleotide probe
  • Said means allow to determine the level of the at least one miRNA in a biological sample isolated from an individual and, thus, to diagnose whether the individual suffers from SIRS or not, to determine the severity of SIRS in the
  • the at least one polynucleotide may be part of a
  • microarray/biochip or may be attached to beads of a beads- based multiplex system.
  • the kit may further comprise (iii) a container, and/or
  • the data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD) , a digital versatile disk (DVD) , a microchip or another semiconductor-based electronical data carrier.
  • the access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized
  • the access code may also allow access to an
  • application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.
  • Said data carrier may further comprise the at least one reference, e.g. the reference level of the level of the at least one miRNA determined herein.
  • the data carrier comprises an access code which allows the access to a database
  • said at least one reference e.g. said reference level may be deposited in this database.
  • the data carrier may also comprise information or instructions on how to carry out the methods according to the first to third aspect of the present invention.
  • Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer (s), a reagent (s) and/or a diluent (s) for determining the level mentioned above.
  • Figure 1 It presents the effects of the surgery and a developing disease as consequence for the controls, SIRS and sepsis patients. miRNAs that are affected in controls are caused by the surgery but not by a developing SIRS or sepsis.
  • the markers of class 1 (only controls) are markers to rule out diseases.
  • the markers of classes 2 and 3 are positive examples where effects also in SIRS / sepsis are observed.
  • the markers of class 3 allow to distinguish between SIRS with infection (sepsis) and SIRS without infection.
  • the miRNA markers shown in Figure 1 allow to determine whether a patient suffers from a systemic
  • SIRS inflammatory response syndrome
  • Figure 2 It shows significant markers in the comparison controls versus SIRS / sepsis with median concentrations, p- values and fold changes. These miRNA markers allow to
  • SIRS systemic inflammatory response syndrome
  • Figure 3 It shows an overview of the identified and claimed miRNA biomarkers with their nucleic acid sequences.
  • Figure 4 It is a Box-plot representation. It shows as an example one miRNA, namely hsa-miR-199a-5p (SEQ ID NO: 24), that is only up-regulated in sepsis patients following surgery and development of the sepsis.
  • Figure 5 It is a Box-plot representation. It shows as an example one miRNA, namely hsa-miR-338-3p (SEQ ID NO: 25), that is only up-regulated in sepsis patients following surgery and development of the sepsis.
  • Example 1 Identification of miRNAs as biomarkers for the diagnosis of a systemic inflammatory response syndrome (SIRS) in a patient
  • RNA including miRNA
  • RNA was synthesized by Qiagen GmbH, Hilden, Germany. Quantification of purified RNA was performed with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) . The quality and integrity of the RNA (RIN value) was
  • Agilent in accordance with the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA) . After rotating hybridization for 20 hours at 55 C, the slides were washed twice and scanned on Agilent's
  • the profiled samples were subjected to normalization.
  • the 2,549 human miRNAs available on the Agilent miRBase v21 arrays were then used for the bioinformatics analysis.
  • different methods were applied (e.g. unsupervised clustering or analysis of variance) .
  • the t-test was used for comparisons between the control group and the other classes.
  • multiple comparison was also carried out using the analysis of variance (ANOVA) test. All P values
  • AUC characteristics curve
  • SIRS SIRS and sepsis
  • SIRS sepsis
  • SIRS sepsis
  • a paired analysis of the respective groups allowed the identification of miRNA biomarkers that changed in time because of the surgical intervention (dysregulated in all groups, especially the controls) and represent exclusion markers (denoted in the following as class 1 markers) , that are specific for SIRS or sepsis but not in controls (referred to as class 2 markers) and that distinguished between SIRS and sepsis (referred to as class 3
  • RNA Prior to RNA extraction, PAXgene tubes were thawed overnight at room temperature to ensure complete lysis of blood cells. Total RNA, including miRNA, was
  • RNA samples were extracted and purified using the PAXgene Blood miRNA Kit in accordance with the manufacturer' s instructions (Qiagen GmbH, Hilden, Germany) . Quantification of purified RNA was performed with NanoDrop 1000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) . The quality and integrity of the RNA (RIN value) was evaluated using Agilent Bioanalyzer and the Nano RNA Kit in accordance with the manufacturer' s protocols (Agilent Technologies, Santa Clara, California, USA) . Sample measurement
  • miRNA expression profiling samples were analysed on Agilent Sureprint G3 Human miRNA (8> ⁇ 60k) microarray slides with the latest miRBase v21 content. Each array targets 2,549 microRNAs with 20 replicates per probe. Extracted miRNA was labeled and hybridized using the miRNA Complete Labeling and Hybridization Kit from Agilent Sureprint G3 Human miRNA (8> ⁇ 60k) microarray slides with the latest miRBase v21 content. Each array targets 2,549 microRNAs with 20 replicates per probe. Extracted miRNA was labeled and hybridized using the miRNA Complete Labeling and Hybridization Kit from
  • Agilent in accordance with the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA) . After rotating hybridization for 20 hours at 55 C, the slides were washed twice and scanned on Agilent's
  • the profiled samples were subjected to normalization.
  • the 2,549 human miRNAs available on the Agilent miRBase v21 arrays were then used for the bioinformatics analysis.
  • different methods were applied (e.g. unsupervised clustering or analysis of variance) .
  • the t-test was used for comparisons between the control group and the other classes.
  • multiple comparison was also carried out using the analysis of variance (ANOVA) test. All P values
  • Such miRNA biomarkers can be used to decide whether an intervention is postponed (in a non-acute care setting) or at least whether patients get special attention in an acute care seeing on an ICU.

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Abstract

La présente invention concerne un procédé de diagnostic d'un individu présentant/souffrant ou non d'un syndrome de réponse inflammatoire systémique (SIRS). En outre, la présente invention concerne un procédé de détermination de la gravité d'un syndrome de réponse inflammatoire systémique (SIRS) chez un individu. En outre, la présente invention concerne un procédé de pronostic d'un syndrome de réponse inflammatoire systémique (SIRS) chez un individu.
PCT/EP2018/064162 2017-05-30 2018-05-30 Miarn en tant que biomarqueurs pour un syndrome de réponse inflammatoire systémique Ceased WO2018219998A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024008950A1 (fr) * 2022-07-08 2024-01-11 Ospedale San Raffaele S.R.L. Cassettes transgéniques

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010088668A2 (fr) * 2009-02-02 2010-08-05 Cepheid Méthodes de détection d'une sepsie
WO2012020308A2 (fr) * 2010-08-13 2012-02-16 The University Court Of The University Of Glasgow Thérapies cellulaires et moléculaires
WO2013040379A1 (fr) * 2011-09-16 2013-03-21 Cepheid Procédés de détection d'une sepsie
WO2015117205A1 (fr) * 2014-02-06 2015-08-13 Immunexpress Pty Ltd Méthode de signature de biomarqueur, et appareil et kits associés
WO2017037477A1 (fr) * 2015-09-04 2017-03-09 The University Of Sussex Marqueur biologique de sepsis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010088668A2 (fr) * 2009-02-02 2010-08-05 Cepheid Méthodes de détection d'une sepsie
WO2012020308A2 (fr) * 2010-08-13 2012-02-16 The University Court Of The University Of Glasgow Thérapies cellulaires et moléculaires
WO2013040379A1 (fr) * 2011-09-16 2013-03-21 Cepheid Procédés de détection d'une sepsie
WO2015117205A1 (fr) * 2014-02-06 2015-08-13 Immunexpress Pty Ltd Méthode de signature de biomarqueur, et appareil et kits associés
WO2017037477A1 (fr) * 2015-09-04 2017-03-09 The University Of Sussex Marqueur biologique de sepsis

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Helvetica Chimica Acta", 1995, article "A multilingual glossary of biotechnological terms: (IUPAC Recommendations"
BACKES Y. ET AL.: "Usefulness of suPAR as a biological marker in patients with systemic inflammation or infection: a systematic review", INTENSIVE CARE MED, vol. 38, 2012, pages 1418 - 1428, XP035101035, DOI: doi:10.1007/s00134-012-2613-1
CHA CHEN: "Differential expression of plasma miR-146a in sepsis patients compared with non-sepsis-SIRS patients", EXPERIMENTAL AND THERAPEUTIC MEDICINE, 30 January 2013 (2013-01-30), GR, XP055316969, ISSN: 1792-0981, DOI: 10.3892/etm.2013.937 *
JIA-FENG WANG ET AL: "Serum miR-146a and miR-223 as potential new biomarkers for sepsis", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 394, no. 1, 26 March 2010 (2010-03-26), pages 184 - 188, XP055004041, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2010.02.145 *
JING XU ET AL: "Serum miR-195 is a diagnostic and prognostic marker for osteosarcoma", INT J CLIN EXP MED, 20 March 2017 (2017-03-20), United States, pages 4756 - 4763510, XP055494285, ISSN: 1940-5901, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0022480414010610?via%3Dihub> DOI: 10.1016/j.jss.2014.11.025 *
STEFANO CASERTA ET AL: "Circulating Plasma microRNAs can differentiate Human Sepsis and Systemic Inflammatory Response Syndrome (SIRS)", SCIENTIFIC REPORTS, vol. 6, no. 1, 20 June 2016 (2016-06-20), XP055494287, DOI: 10.1038/srep28006 *
TIHOMIR ILIEV VACHEV: "Down regulation of miRNA let-7b-3p and let-7d-3p in the peripheral blood of children with autism spectrum disorder", INT.J.CURR.MICROBIOL.APP.SCI, vol. 2, 1 December 2013 (2013-12-01), pages 384 - 388, XP055494321, Retrieved from the Internet <URL:https://www.ijcmas.com/vol-2-12/Tihomir%20Iliev%20Vachev,%20et%20al.pdf> [retrieved on 20180720] *
WACKER C. ET AL.: "Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis", LANCET INFECT DIS, vol. 13, 2013, pages 426 - 435
XUE ZHAO ET AL: "The predict value of serum miRNA150 on sepsis prognosis", INT J CLIN EXP MED, 30 September 2016 (2016-09-30), England, pages 518367 - 18372, XP055494297, Retrieved from the Internet <URL:https://www.researchgate.net/publication/309076587_The_predict_value_of_serum_miRNA150_on_sepsis_prognosis> [retrieved on 20180720] *
YUQIAN MA ET AL: "Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis", PLOS ONE, vol. 8, no. 10, 16 October 2013 (2013-10-16), pages e75918, XP055390796, DOI: 10.1371/journal.pone.0075918 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024008950A1 (fr) * 2022-07-08 2024-01-11 Ospedale San Raffaele S.R.L. Cassettes transgéniques

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